Polymerase chain reaction (PCR) is a primer extension reaction that provides a method for amplifying specific nucleic acids in vitro. Widely used for cloning and other molecular biological manipulations (Mullis et al., 1987, Methods Enzymol.; Saiki et al., 1985, Science), the method can produce a million to a billion fold copies of a DNA template in a single enzymatic reaction within a matter of minutes to hours, enabling researchers to determine the size and sequence of a target DNA.
Despite significant progress, improvements in several areas of PCR technology are still needed. One particular area concerns non-specific DNA amplification due to mis-priming. During a PCR reaction, the particular stretch of DNA to be amplified, called the target sequence, is identified by a specific pair of oligonucleotides called primers. The specificity of DNA amplification typically depends on the specificity of primer-target hybridization, which is usually reflected by the melting points, Tm, of the primer-target hybrids and is also affected by the temperature at which the primer extension reaction takes place. In general, more specific primer-target hybridization occurs at higher temperatures, typically 50-70° C. Since the chain extension step of a PCR reaction is usually carried out within this temperature range, during which Taq is also most active, nonspecific DNA amplifications are mostly minimized. On the other hand, so-called mis-priming, which results from primer hybridizing to partially complimentary sequences or to other primers, takes place more frequently at lower temperatures such as room temperature. Another problem is that Taq still has significant residual activity at room temperature although the enzyme exhibits the highest activity in the normal PCR temperature range of 50-70° C. The partially or weakly hybridized primers coupled with the residual Taq activity at a lower temperature such as room temperature could lead to significant formation of nonspecific amplification products. When the temperature is elevated to 50-70° C. to initiate a PCR reaction, these non-specific products formed at the lower temperature sometimes now act as templates and compete with the amplification of the desired product, thus leading to even greater amount of nonspecifically amplified products. Since a PCR reaction is usually first assembled at room temperature, it is therefore highly desirable and in some cases essential to suppress any polymerization activity at lower temperatures such as room temperature before the desired amplification reaction takes place at higher temperatures.
To overcome the aforementioned problem, various so-called “hot-start” PCR methods have been developed that allow one to turn off the amplification reaction at room temperature and to turn on when the temperature is raised to the normal PCR operating temperature range. In general, nearly all known hot-start methods employ a strategy of suppressing the polymerase activity at below the PCR temperature range.
One earlier hot-start method involves withholding one or more components of a PCR reaction until the reaction vessel is heated to the PCR operating temperature (U.S. Pat. No. 6,403,341, Barnes et al., Frohman M A et al, PNAS (1998) 85:8998-9002). Although the method is conceptually straight forward, the extra step required for reagent mixing is not only inconvenient but also susceptible to contamination and incompatible with high throughput operations.
Another earlier version of hot-start method suppresses the polymerase activity by creating a temperature-dependent physical barrier, such as the use of wax, to separate the PCR components (U.S. Pat. Nos. 5,413,924, Horton et al., Biotechniques 16:42-43). As an example, Taq DNA polymerase can be stored in wax beads while the rest of the PCR components are left in the aqueous phase. When the reaction vessel is heated to the denaturing temperature of PCR, usually above 90° C., the wax melts, thus mixing all components. However, since mixing of the components relies largely on heat convection, which is most likely inefficient and incomplete in view of the designs of most of the commercial PCR instruments, this method is again not widely used.
Most of the commercially available hot-start polymerases, or so-called master-mixes that contain all necessary components to start a PCR except for the primers and template, now employ a pre-mixed and single-phased format that avoids manual reagent addition and convection-based reagent mixing.
One strategy of developing a hot-start polymerase is to chemically modify the enzyme, more specifically, the lysine residues of the enzyme, with a thermally unstable chemical group to inactivate the enzyme. Once the reaction is heated to temperatures above 90° C. for 10 to 20 minutes, the heat-labile modifying group is cleaved from the enzyme, thus re-activating the enzyme activity. (U.S. Pat. Nos. 5,677,152 and 6,183,998). There are a couple of major drawbacks with this method. First, since there are multiple lysine residues in the enzyme, the modification reaction is difficult to control, typically resulting in a complex mixture of labeled enzyme with some enzyme molecules more heavily labeled and others lightly labeled. Accordingly, the temperature response of the modified enzyme molecules is often heterogeneous with the lightly labeled enzyme molecules more easily re-activated but the more heavily labeled enzyme molecules more difficult to be reactivated. Second, the yield for making the modified enzyme is relatively low, typically in the range of 10 to 50%, which, when coupled with the relatively high cost of the enzyme itself, can make this method uneconomical. Finally, it is desirable to shorten the 10- to 20-minutes enzyme activation time, which makes up a significant portion of the overall PCR time with this method.
Another hot-start PCR method employs polymerase-specific monoclonal antibodies to inhibit the enzyme activity. Often an antibody to enzyme molar ratio of seven to one is used to sufficiently inactivate the enzyme. The monoclonal antibodies bind to the polymerase at lower temperatures such as room temperature in a manner to inactivate the enzyme. When the reaction temperature is raised above 90° C., the antibodies lose affinity for the enzyme, which therefore becomes reactivated again. (U.S. Pat. No. 5,338,671, Scalice E R et al, Kellogg et al, (1994) Biotechniques 16:1134-1137). One advantage of this method is that the enzyme activation time is only 1 to 3 minutes, a significant improvement over the chemically modified enzyme method. However, a major drawback with this method is the relatively high cost associated with the use of a large amount of antibody molecules.
Still another hot-start PCR method employs negatively charged polymers to block polymerase as disclosed in the US. Pat. Application No. US2003/0092135A1. The negatively charge polymers exhibit temperature-dependent inhibition to Taq DNA polymerase activity with high inhibition at low temperatures and low or no inhibition at high temperatures.
Other variations of negatively charge polymers have also been used to inhibit DNA polymerase activity. For example, short DNA fragments (Kainz P. et al (2000) BioTechniques 28:278-282, also U.S. Pat. No. 6,830,902B1) or aptamers (U.S. Pat. No. 5,693,502) have been used to formulate hot-start PCR reactions. These oligonucleotide-based inhibitors bind to a DNA polymerase by mimicking the natural substrate of the enzyme. At elevated temperatures, the inhibitors lose their binding affinity for the enzyme, rendering polymerase available to its normal substrate. However, in order to achieve complete enzyme inhibition at lower temperatures, the concentration of the inhibitors typically needs to be in the hundreds of micromole range so that they can compete effectively for enzyme binding with the natural substrate. Although the hot-start method using these inhibitors also has the advantage of relatively short enzyme activation time, typically within a minute, the high cost of having to use a large quantity of the inhibitors makes this technology unpractical.
Still there remains a need for a hot-start PCR method that is single-phased, fast to hot-start, low cost and easy to handle all at the same time. As disclosed herein, various aspects of the present invention address these objectives.